As a result of stem cell studies, which have recently been rapidly advanced, it has become possible to separate stromal stem cells from fat tissues and allow the stem cells to proliferate and differentiate into desired cell types. However, there are many problems in the practical application of stem cell culture, a large number of expensive systems and facilities, researcher staffs and expensive suppliers are necessary.
Particularly, when the cell culture process is carried out without thorough preparation, contamination with bacteria in air can occur, leading to the failure of the entire process, and there will be various failure factors, including a failure caused by the mistake of research staffs. For this reason, to obtain successful study results, enormous equipment investment and the training of research staffs are required.
For example, clean rooms or clean benches, in which air flows downward, are required for reducing contamination with bacteria in air. Also, attendant facilities, including disinfection equipment for disinfecting various containers, air cleaners, and suction units, are required. Moreover, research staffs should necessarily be trained through long-term exercise in order for the research staffs to use such facilities correctly and not to be contaminated during complicated processes.
Meanwhile, conditions for inducing cell proliferation include many chemical environments, but some of which are not yet clearly understood. The inhibition of cell proliferation is also greatly influenced by the environment thereof, and in this case, it seems that there are many principles, which are not yet established. For example, there is an experimental report that, when germs are present at a density of more than 106 germs per cm2 of human tissue, the proliferation thereof in the human body will be accelerated to cause inflections, but the precise mechanism thereof is not yet established. In the case of the culture of general cells, there are reports that, when more than a given number of cells are somewhat clustered, cell proliferation signals occur to induce the exponential proliferation of the cells; however, precise materials and principles for the cell proliferation are not yet established, except that the cell proliferation is possible in water-soluble materials and conductive materials.
For the inhibition of cell proliferation, various environments have also been reported. Among them, a clear principle is the contact inhibition phenomenon that, when cells are present attached laterally, the cells no longer proliferate in that direction.
In these points of view, the prior culture methods for cell proliferation have advantages and disadvantages as follows.
[Plate Culture]
A traditional cell culture method is a plate culture method of inducing cell division on a plate in medium. In this method, specific cells are spread and attached to the plate and allowed to grow and proliferate toward their surrounding regions. Then, as the cells reach saturation density, the cells are isolated from the structure having the cells attached thereto, using an enzyme such as trypsin. The resulting cell suspension is centrifuged, and the cells deposited on the bottom of the centrifuge are collected, and suspended in a sufficient amount of phosphate buffer saline (PBS) to wash out the enzyme. The cell suspension is centrifuged again to deposit the cells on the bottom of the centrifuge. After this process is repeated several times, the cells are suspended in medium and seeded (loaded) onto a fresh plate. As the cells grow to saturation, the same process as described is repeated. In this way, an exponential increase in the number of the cells can be achieved.
This method has a limitation on proliferation rate, because cell proliferation occurs only at the periphery of cell colonies, cells surrounded by other cells do not proliferate, and thus substantial cell proliferation occurs only at the outer edges of cell colonies. For this reason, the principle of plate culture is to maximize the number of proliferating cells by increasing the number of cell colonies.
In this case, the cell adhesion surface is the bottom or wall of a container, and only a single cell layer is formed without the deposition of cells. Thus, the plate culture is also called “mono-layer culture”.
[Three-Dimensional Culture in the Absence of Scaffold]
This is a culture method for solving the limitation of the plate culture by controlling proliferation conditions in a state in which cells are suspended in liquid or on gel. In this method, a flow of liquid may also be controlled for proper conditions. Conceptually, this culture method is three-dimensional culture, shows no cell-to-cell contact inhibition and has much more chances than those of the plate culture with respect to the direction of proliferation of cells. However, the recognition of proliferation signals between cells and the division rate of cells are reduced. Thus, this method has limitations on the kind of cells and culture conditions in order to substantially increase proliferation rate.
[Three-Dimensional Culture Using Scaffold Other than Micro-Scaffolds]
A scaffold is a structure on which cells can adhere and grow and is a material, which serves to secure a space for cell growth and, at the same time, control the shape of cell colonies, because the shape of cell colonies depends on the scaffold. Culture using the scaffold is called “three-dimensional culture” in the sense that it has a cell adhesion surface significantly larger than that of the plate culture and that it can form a layered structure, because cells can grow not only in a planar direction, but also in upward and downward directions.
Scaffolds for use in three-dimensional culture are mostly solids, but in some cases, may also be gelatin-like materials which exist in an intermediate state between liquid and solid.
The scaffolds have a porous structure, which allows the movement of most of media and has a maximized surface area for cells adhesion. These scaffolds are in the shape of a sponge-like disc, a block or a sphere.
These scaffolds have a structure in which cell adhesion surfaces are layered or curved into a complicated shape so as to maximize the cell adhesion surface area. However, because cells can grow through the scaffolds, they can hardly grow inside of the scaffold, and thus reach saturation. For this reason, the cells can further proliferate only after a process of separating the cells from the scaffold after saturation and seeding the separated cells onto a fresh scaffold. Thus, a process of separating and washing cells are necessarily performed as in the plate culture.
[Three-Dimensional Culture Using Micro-Scaffolds]
Although this culture method employs solid powder, it is considered that the powder particles are saturated while they aggregate together by cells. For cell dispersion and seeding for increasing the proliferation of cells, a process of separating cells using an enzyme such as trypsin should generally be performed as before. This culture method shows an increase in proliferation rate and efficiency compared to other prior methods, but has problems in that the cell separation and seeding step still requires a given amount of effort and time, and cell viability is reduced due to chemical separation.
[Suspension Culture Using Scaffold]
In this culture method, cells together with a given amount of scaffolds are continuously suspended to induce cell adhesion to the scaffolds. However, as the scaffold particles are saturated due to the adhesion of cells, the cells no longer proliferate. Thus, the number of particles, on which cell proliferation occurs, increases with the passage of time, but the rate of saturated particles also increases, leading to a decrease in the total amount of proliferation inhibition. In this method, it is easy to maintain cells at constant density by regulating only media; however, because this method is dynamic, the proliferation-inducing signal exchange between cells does not occur and there is no advantage in a state in which cells adhere to a static surface.
[Static Culture Using Scaffold]
An example of static culture is a method comprising seeding cells onto a sufficient amount of scaffolds and forming a vortex flow in the medium to move non-adhered cells so as to increase the chance to adhere to the scaffolds. However, cells are partially saturated to increase the ratio of cells surrounded by the same cells, thus increasing the amount of cell-to-cell contact inhibition and reducing the amount of cell proliferation signals due to an excessively low cell density in an initial state.
[Initial Shaking and Static Culture Using Scaffold]
In this culture method, the amount of cell-to-cell contact inhibition can be minimized by seeding cells onto a sufficient amount of scaffolds and shaking the medium at a suitable point of time only in an initial stage. However, the amount of proliferation-inducing signals between cells is also small due to an excessively low cell density in the initial stage. Also, cells are chemically separated, a process of adding an exponential number of scaffolds is not adopted, and thus a chemical cell separation process is performed for dispersion and seeding onto fresh scaffolds having no cells. However, when the addition of an exponential amount of scaffolds is not performed, a large amount of scaffolds should be used in an initial stage. For this reason, in this method, the penetration of medium into the scaffolds can become difficult from the initial culture stage to reduce the efficiency of the initial culture stage, thus making it difficult to maintain stem cells.